Methods of Selecting and Using Therapeutic and Prophylactic Probiotic Cultures to Reduce Bacterial Pathogen Loads

ABSTRACT

Methods are provided for selecting a bacterium capable of reducing pathogenic bacterial colonization of the intestinal tract in a subject comprising selecting the bacterium capable of migrating at least 0.75 cm from the point of inoculation on motility agar after incubation for 24 hours at 37° C. or capable of migrating from the point of inoculation to a diameter of at least 1.5 cm based on the farthest colonies from the point of inoculation on motility agar after incubation for 24 hours at 37° C. Bacteria selected using the method and compositions comprising these bacteria are also provided.

BACKGROUND

Campylobacter is one of the most commonly reported bacterial causes ofhuman food-borne illness in the United States and epidemiologicalevidence indicates poultry and poultry products as a significant sourceof human Campylobacter infection. Colonization of poultry byCampylobacter is widespread and difficult to prevent even with properbiosecurity measures. Campylobacter is considered a commensal organismin avian species and it is predominantly found in the lowergastrointestinal tract of birds, concentrated in the mucus layer of thececal crypts, cloaca and large intestine. The evisceration techniques atthe processing plant are usually the most common source of carcasscontamination with Campylobacter.

Reducing Campylobacter in the intestinal tract would reducecontamination of poultry products. A variety of approaches for reducingthe colonization of Campylobacter in poultry have been explored, butwith varying degrees of success. These include use of enzymes such asxylanase, bacteriophage, bacteriocins, frutooligosaccharides and mucinutilizing coliforms, organic acids and antibiotics.

Another approach to decrease food-borne pathogens and minimize the useof antibiotics is by the use of competitive exclusion (CE) microflora.Also known as probiotics, CE was first introduced by Nurmi and Rantala(Nature (1973) 241:210-211). Probiotics are composed of single ormixtures of selected non-pathogenic organisms that upon ingestion havethe ability to colonize the gastrointestinal tract and improve thehosts' health. Probiotics are usually given orally at day-of-hatch/birthallowing these bacteria to first colonize the intestinal tract beforethe bird/animal is exposed to pathogenic microorganisms present in theenvironment. The probiotic cultures used for poultry are generallyobtained from intestinal tracts of healthy young birds. Undefinedprobiotic cultures cannot assure the absence of pathogenic organisms,guarantee the same strains are present for every treatment; and are notapproved by regulatory agencies for use in the U.S. Nonetheless, the“Nurmi concept” has been demonstrated to be an effective means inreducing Salmonella colonization in broilers. Although the results todate are promising against Salmonella, Campylobacter has presented amore difficult target.

SUMMARY

Methods of selecting bacterial strains capable of inhibiting the growthor colonization capability of pathogenic bacteria and several of theselected bacterial strains are provided herein. Methods of using theseselected strains are also provided and several strains selected by themethods are provided.

In one aspect, methods of selecting a first bacterium capable ofreducing bacterial colonization, in particular pathogenic bacterialcolonization, of the intestinal tract of a subject are provided. Themethods include selecting bacteria with enhanced motility relative tocontrols of the same species. In particular, bacteria capable ofreducing pathogenic bacterial colonization of the intestinal tract in asubject by selecting the first bacterium capable of migrating at least0.75 cm from the point of inoculation on motility agar after incubationfor 24 hours at 37° C. or capable of migrating from the point ofinoculation to a diameter of at least 1.5 cm based on the farthestmigrating bacterial colonies from the point of inoculation on motilityagar after incubation for 24 hours at 37° C. are selected. The bacteriamay be further selected to include those bacteria Generally Regarded asSafe by the Food and Drug Administration.

In another aspect, the bacteria selected by the methods described hereinand compositions comprising the bacteria are described.

In yet another aspect, isolated Bacillus subtilis strains designated asME-1, ME-2 and ME-3 are provided. Compositions, such as probiotic foodproducts or nutraceuticals, comprising the isolated Bacillus subtilisstrains or other bacterial isolates selected by the methods are alsoprovided.

In still another aspect, methods of inhibiting growth of pathogenicbacteria are provided. The methods include delivering a bacteriumselected by the methods described herein and having enhanced motility orat least one of the Bacillus subtilis strains ME-1, ME-2 or ME-3, acombination thereof or a composition comprising at least one of thebacterium selected by the methods described herein or one of theBacillus subtilis strains (ME-1, ME-2 or ME-3) to a target region.

In a still further aspect, methods of reducing pathogenic bacterialcolonization in a subject are provided. The methods include oraladministration of a bacterium selected by the methods described hereinand having enhanced motility or at least one of the Bacillus subtilisstrains ME-1, ME-2 or ME-3, a combination thereof or a compositioncomprising at least one of the bacterium selected by the methoddescribed herein or one of the Bacillus subtilis strains (ME-1, ME-2 orME-3) to the subject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the diameter of spread after each subsequentselection and passage for motility enhancement of the bacterialisolates.

FIG. 2 is a graph showing the concentration of Campylobacter per gram ofcecal contents seven days after inoculation with Campylobacter in twoweek old chicks that were either not treated with a probiotic or weretreated on the day of hatch with the indicated isolates or combinationsof isolates. Three separate trials are shown and columns within the sametrial with different superscripts denote significant difference(P<0.05).

FIG. 3 is a graph showing the concentration of Campylobacter per gram ofcecal contents .seven days after inoculation with Campylobacter in twoweek, old chicks. The chicks were either not treated with a probiotic(control) or were treated on the day of hatch with the original isolate1 or with the motility enhanced isolate 1. Three separate trials areshown and columns within the same trial with different superscriptsdenote significant difference (P<0.05).

DETAILED DESCRIPTION

Campylobacter is a pathogenic bacterium that is a leading cause offood-borne illness. Campylobacter infection is often associated with theconsumption of poultry products. Campylobacter is commonly present inthe intestinal tract of poultry and one strategy to reduce entericcolonization is the use of probiotic cultures. Such probiotic cultureshave been developed and shown to be effective to reduce colonization ofSalmonella, but the results for Campylobacter have not been, promising.

As described in the Examples, we discovered that selection of bacteriafor enhanced motility resulted in selection of bacteria capable ofreducing colonization of other pathogenic bacteria in a subject such asCampylobacter. The isolated bacteria were selected based on initialmotility and were then further selected by serial passage on motilityagar selecting the most motile isolates to select for enhanced motility.Those bacteria with motility characteristics that may be useful toreduce colonization by pathogenic bacteria in a subject were bacteriacapable of migrating at least 0.75 cm, 1 cm, 1.5 cm or even 2 cm fromthe point of inoculation on motility agar after incubation for 24 hoursat 37° C. or capable of migrating from the point of inoculation to adiameter (to form a zone of migration with a diameter) of at least 1.5cm, 2 cm, 3 cm, or even 4 cm based on the farthest migrating bacterialcolonies from the point of inoculation on motility agar after incubationfor 24 hours at 37° C. are selected. Motility agar from Becton-Dickinsonwas used in the Examples. Those skilled in the art will appreciate thatother bacterial motility media are available and can be used to developsimilar assays.

As described in the Examples, three strains of Bacillus subtilis wereselected for enhanced motility and were demonstrated to inhibitCampylobacter growth in vitro and in vivo in chickens. The three strainsare designated ME-1, ME-2 and ME-3 (ME: motility enhanced). These threestrains or other similar isolates selected using the methods disclosedherein may be used to make probiotic compositions that contain at leastone of the isolates or any combination thereof. The compositions mayalso contain bacterial strains in addition to those disclosed herein.The compositions may be used in food products, as additives to drinkingwater or in nutaiceuticals or pharmaceuticals. Thus, food products,additives and nutraceuticals including at least one of ME-1, ME-2 orME-3 or any other bacteria selected using the method of selectiondescribed herein are provided.

As described in the Examples, the three strains were selected byharvesting bacteria from the ceca of two week old chickens and thenidentifying those isolates Generally Recognized as Safe (GRAS) based onthe Food and Drug Administration (FDA) list; identifying which of theseGRAS isolates demonstrated motility characteristics (6 isolates); andfinally determining which of the motile, GRAS isolates were capable ofinhibiting Campylobacter growth in vitro (5 isolates). The five isolateswere then subjected to serial passage and selection for new isolateswith the greatest motility. Three of the isolates demonstrated improvedmotility with serial passage and selection and these three isolates arenow designated as ME-1, ME-2 and ME-3.

Thus a novel method for selection and isolation of bacterial strainscapable of inhibiting growth and/or colonization of pathogenic bacteriain the gastrointestinal tract of a subject, such as Campylobacter invitro and in vivo, is provided. The pathogenic bacteria include bacteriathat are capable of causing disease, e.g., morbidity or mortality, inhumans. These pathogenic bacteria may cause no significant disease inthe host or subject being treated or administered the probioticcompositions. For example, Campylobacter and Salmonella are commensalorganisms in chickens and generally do not cause disease in chickens,but both represent major sources of food-borne illness in humans andthus are considered pathogenic organisms herein. Pathogenic organismsalso include organisms that cause disease in the host or subject beingtreated or administered the composition comprising the bacteria selectedusing the methods described herein or any of the three isolatesprovided, namely ME-1, ME-2 or ME-3.

The bacteria selected for their ability to inhibit the growth orcolonization of pathogenic bacteria may be from any genera of bacteria.The selected bacteria are suitably enteric bacteria that are part of thenormal gut flora of the animal or of the subject being treated. As notedabove, the selected bacteria may also be normal gut floral of a human.Suitably, the selected bacteria are further selected to include GRASbacteria. Suitably, the selected bacteria are from genera such asEscherichia, Salmonella, Shigella, Bacillus, Clostridium andBacteriodes.

The methods include selecting a bacterium with enhanced motilityrelative to an unselected bacterium of the same species. Suitably thebacteria may be selected for enhanced motility relative to bacteria inthe same culture or even from the same isolate via serial passage andselection of bacteria from the culture or isolate that have the greatestmotility from the point of inoculation. The bacteria may be selected bythe ability to migrate a particular distance from an inoculation pointor to produce a zone of migration having a certain diameter. Suitably,the selected bacteria are capable of migrating from the inoculationpoint at least 0.5 cm, more suitably the distance is at least 0.75 cm,0,9 cm. 1.0 cm, 1.2 cm, 1.4 cm, 1.6 cm, 1.8 cm or 2.0 cm by 24 hoursafter inoculation when incubated at 37° C. on a motility agar such asBBL Motility medium from Becton-Dickinson. The bacteria may be selectedby the diameter of the zone of migration as an alternative. Suitably,the selected bacteria are capable of migrating such that the thediameter of the zone of migration is at least 1.0 cm, 1.2 cm, 1.4 cm,1.6 cm, 1.8 cm, 2.0 cm, 2.2 cm, 2.5 cm, 2.7 cm, 3.0 cm, 3.2 cm, 3.5 cm,3.7 cm, or 4.0 cm by 24 hours after inoculation when incubated at 37° C.on a motility agar such as BBL Motility medium from Becton-Dickinson.

The bacteria capable of reducing pathogenic bacterial colonization ofthe intestinal tract of a subject may also be selected using additionalcriteria. The bacteria may be further selected using the FDA GRASclassification scheme, such that only bacteria approved as GRAS areselected. Those of skill in the art will appreciate that bacterialcharacterization maybe accomplished using commercially availablebacterial typing technology such as the Biolog system used in theexamples or may be accomplished using traditional bacterial typingtechniques including microscopic characterization using stains,differential growth or colorimetric staining on different growth mediaor other PCR or DNA typing based methodologies.

The bacteria may be further selected for their ability to inhibit thegrowth of pathogenic bacteria, such as Campylobacter, Salmonella, or E.coli, in vitro. The ability to inhibit the growth of pathogenic bacteriamay be measured in a variety of ways, one of which is described in theExamples. The ability of the bacteria to create a zone of inhibition ofgrowth of the pathogenic bacteria when plated in discreet locations on aplate confluent for the pathogen is one way of measuring the ability toinhibit growth of a pathogen in vitro. Those of skill in the art willappreciate that similar assays can be performed in solution.

The bacteria may also be selected for their ability to reducecolonization of pathogenic bacteria in a subject. The pathogenicbacteria may be Salmonella, E. coli or Campylobacter. The subjects maybe poultry (including chicken and turkeys), cattle, pigs, mice, rats,humans or other domesticated animals or pets such as dogs, cats,lizards, snakes. In the Examples, ME-1, ME-2 and ME-3 were tested fortheir ability to reduce Campylobacter colonization in chickens. Themotility enhanced bacterial isolates were orally administered (by oralgavage) to day of hatch chicks. Two weeks later the chicks wereadministered a mixture of Campylobacter isolates. After an additionaltwo weeks the chicks were sacrificed, ceca harvested and Campylobacterconcentration in the ceca determined. The motility enhanced bacteriawere capable of reducing the colonization of the chicken ceca withCampylobacter. Similar results are expected with other pathogenicbacteria such as Salmonella and E. coli.

The methods of selecting bacteria capable of reducing bacterialcolonization of the intestinal tract described herein may be readilyapplied by those of skill in the art to isolate other bacteria capableof reducing colonization by pathogenic bacteria. For Example, bacteriamay be harvested from the intestinal tract of cattle and selected forenhanced motility as described herein to select for bacteria capable ofreducing pathogenic bacterial colonization of cattle. The pathogenicbacteria may be Salmonella or E. coli as well. Alternatively, bacteriamay be selected from one species and used to treat another. For example,ME-1 was selected from a chicken but may be used to treat an animalother than a chicken such as a turkey or cow.

Compositions comprising the bacteria selected using the methodsdescribed above are provided. The compositions may include apharmaceutically acceptable carrier, diluent and/or excipient. Thecomposition may include more than one bacterial isolate selected by themethods described herein or may also include other bacterial isolates.The compositions may be formulated for delivery in food, water, via oralgavage or via an aerosol or sprayable product.

Methods of inhibiting growth of pathogenic bacteria by delivering acomposition including a bacteria selected by the methods describedherein or at least one of the strains described herein (ME-1, ME-2 orME-3) or a composition comprising at least one of the strains to atarget region are provided. The strains may be delivered singly or incombination with other strains including those described herein andthose not disclosed herein or selected using other methods. The targetregion may be within a solution, in an area, on a surface, an object ora subject suspected of being contaminated with, harboring or beingsusceptible to contamination with a pathogenic bacterium.

Delivery of a composition containing a bacterium selected by the methodsdescribed herein or one or more of the described bacterial isolates,ME-1, ME-2 or ME-3 allows the growth of the pathogenic bacteria to beinhibited or reduced as compared to a control. A suitable control wouldbe a similar target region to which the bacterial strains disclosedherein or a composition comprising the ME-1, ME-2 or ME-3 strains werenot delivered. The bacteria described herein may actively inhibitpathogenic bacterial growth, but need not do so. The reduction orinhibition of growth of or colonization by the pathogenic bacteria maybe accomplished through any other means, including out-competing thepathogenic bacteria for the niche in which the bacteria colonize and/orgrow in the subject. The growth of the pathogenic bacteria may beinhibited or reduced as compared to untreated controls by 2-fold,5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold oreven by as much as 2 logs, 3 logs or 4 logs. Growth inhibition may bemeasured by any means, including those known to those of skill in theart, such as plating for colony forming units, real-time PCR, andspectroscopic determination.

The strains or compositions may be delivered to the target region usinga variety of methods. Suitably, the bacterial strains or compositionscomprising them are added directly to the target region. Delivery mayinclude mixing, direct contact, or may rely on processes such asdiffusion. Alternatively, the strains or compositions may be mixed witha solution prior to delivery to the target region to facilitate thedelivery. The compositions may be used to treat subjects but may also beused to treat surfaces or objects that subjects may come into contactwith and form a source of potential infection by pathogens.

Methods of reducing pathogenic bacterial contamination in thegastrointestinal tract of a subject are also provided. These methodsinclude orally administering a bacteria selected using the methodsdescribed herein or at least one of ME-1, ME-2, ME-3 or a compositioncomprising at least one of these strains to the subject. The subjectsinclude but are not limited to poultry such as chickens and turkeys,cattle, humans, pigs, mice, rats, cats, dogs or other domesticatedanimals or pets. As noted above the pathogenic bacteria may include, butare not limited to Campylobacter, Salmonella and E. coli.

The strains or compositions described herein may be administered in avariety of ways known or available to those skilled in the art. Thestrains or compositions may be administered via oral gavage as describedin the examples, or alternatively may be administered in the form of apharmaceutical, nutraceutical, added to the food or water or provided inaerosolized or sprayable form for administration by inhalation. Inaddition, the strains and compositions described herein may be providedas liquid suspensions, lyophilized or freeze dried powders or frozenconcentrates for addition to target regions other than a subject.

The strains or compositions are capable of reducing colonization ofpathogenic bacteria in the gastrointestinal tract of the subject. Thegastrointestinal tract includes any portion of the gastrointestinaltract. For example, the method may result in a reduction of pathogenicbacterial colonization of the mouth and thus prevent the formation ofcavities. Alternatively, the methods may reduce pathogenic bacterialcolonization of a portion of the gastrointestinal tract, such as thececa or ileum of a chicken. In the Examples, the bacterial strains wereshown to reduce the colonization of Campylobacter in the ceca ofchickens.

Reductions in pathogenic bacterial colonization may be determined bycomparing the concentration, i.e. the number or colony forming units, ofthe pathogenic bacteria in a treated subject to that in a controlsubject that was not administered the strains or compositions describedherein. The concentration of the pathogenic bacteria in thegastrointestinal tract (or in a portion of the gastrointestinal tract)of the subject may be determined using a variety of assays available tothose skilled in the art. The portion of the gastrointestinal tract ofinterest may be harvested from the animal, washed or scraped to collectbacteria and then the concentration of bacteria determined usingtechniques such as differential plating and colony forming unitanalysis, real-time PCR for the pathogenic bacteria, microscopicdetermination using stains or dyes specific for the pathogenic bacteriaor other assays.

The methods may result in a significant reduction in the pathogenicbacterial colonization of the subject as compared to control subjects.Suitably pathogenic bacterial colonization is reduced by at least 15%,20%, 25%, 30%, 35%, 40%, 45%, 50% or even more. Pathogenic bacterialcolonization may be reduced by 2-fold, 3-fold, 5-fold, 10-fold, 15-fold,or even 20-fold. The pathogenic bacterial load may be reduced by 1 log,2 logs, 3 logs, 4 logs or even 5 logs as compared to the pathogenicbacterial load in a control subject not treated with at least one of theME-1, ME-2 or ME-3 bacterial strains or another enhanced motility strainselected by the methods described herein or a composition comprising atleast one of these strains. The method may be used prophylactically toprevent, infection or colonization with a pathogenic bacterium.Prophylactic administration requires that at least one of the ME-1, ME-2or ME-3 bacterial strains or another enhanced motility strain selectedby the methods described herein or a composition comprising at least oneof these strains is administered to the subject prior to the subject'scolonization with the pathogenic bacteria.

Alternatively the method may be used to decrease bacterial load aftercolonization or infection with pathogenic bacteria. A variety of methodsmay be used to determine if a subject has been exposed to or iscolonized with a pathogenic bacterium. For example, an immunoassay ofthe subject's blood may demonstrate prior exposure to a bacterialpathogen by the presence and ability to detect antibodies directedagainst the pathogen. The subject's saliva or feces may also be testedfor the presence of the pathogenic bacteria prior to initiation of themethods. In the Examples, the chicks were administered the bacterialstrains prophylactically on the day of hatch, such that prior exposureto the pathogenic bacterium was minimized.

The following examples are meant to be illustrative of the invention asdescribed and claimed and are not meant to limit the scope of theinvention. All references cited herein are hereby incorporated byreference in their entireties.

EXAMPLES Materials and Methods Isolation Studies Probiotic CollectionStudy

Day-old broiler chicks (n=80) were obtained from a commercial hatcheryand placed into floor pens with dimensions of 3.06 m² (33 ft²)previously equipped with fresh litter (pine shavings) and heaters. Birdsin this and subsequent trials had ad libitum access to both feed (dietformulated to meet or exceed the requirements of the NEC) and waterduring all trials. At 2 weeks of age the birds were euthanized using CO₂and ceca collected.

Identification and Selection of Isolates

Euthanized chicks were placed on a surgical board, the abdominalfeathers were removed, and the skin was disinfected with 70% alcohol. A5 cm incision was made on the abdominal skin and the ceca were removed.The ceca from each bird was placed into a sterile bag and individuallystored at −80° C. (VWR Forma Scientific, Inc, Ohio) until analysis. Thececa were thawed and the contents were squeezed into sterile tubes anddiluted with Butterfield's Phosphate Diluent (BPD—Difco, BectonDickison, Md.) in three ten-fold dilutions. One hundred μL of eachsolution was spread on Blood Agar Plates (BAP—Difco, Becton Dickison,Md.), and the plates were aerobically incubated for 24 h at 37° C.,Isolated colonies were picked and streaked onto Tryptic soy agar (TSA;EMD, N.J.) to assure purity and incubated aerobically at 37° C. for 24h. Single, isolated colonies were individually grown in 5 mL of TrypticSoy Broth (TSB—Difco, Becton Dickison, Md.) for 8 h or until turbid.Bacteria were identified using the Biolog® system (Biolog, inc.,Hayward, Calif.; Holmes et al., J. Clin. Microbiol. (1994) 32(8):1970-1975; Kersters et al., 1997, Utility of the Biolog system for thecharacterization of heterotrophic microbial communities. 20:3, 439-447),Stocks of those bacteria were prepared by centrifuging TSB at 3000 rpmfor 10 rain, pouring off supernatant and resuspending in 2 mL TSB with20% filter-sterilized glycerol. The suspensions then were dispensed into1 mL aliquots and stored at −80° C.

Further selection of isolates was conducted by using three criteria: 1)identifying those isolates Generally Recognized as Safe (GRAS) based onthe FDA list; 2) identifying which of these GRAS isolates demonstratedmotility characteristics; and 3) determining which of the motile, GRASisolates were capable of inhibiting Campylobacter growth in vitro. Todetermine motility characteristics GRAS isolates were inoculated inSulfide Indole Motility (BBL™ SIM medium, Becton-Dickinson) in a steriletube and incubated for 24 hours at 37° C. The bacteria were consideredmotile if migration away from the line of inoculation occurred. Thosebacteria positive for motility were further tested in vitro againstCampylobacter using the soft agar overlay technique to detectantimicrobial activity (Fredericq, Annu. Rev. Microbiol (1957) IT7-22;Miyamoto et at., Poult. Sci. (2000) 79:7-11 and Zhang et al., J. FoodProt. (2007) 70(4):867-873). The selected isolates were tested againstfour strains of wild type Campylobacter jejuni isolated from chickensand one ATTC strain as previously described by this laboratory (Farneli.et al., J. Appl. Microbiol. (2005) 99:1043-1050). Briefly, 10 μL loopsof each frozen strain were cultured into 5 mL Campylobacter enrichmentbroth (CEB; International Diagnostics Group, Lancaster, England) andincubated for 48 h at 42° C. For the second passage, again 10 μL loopsfrom each strain were transferred into fresh 5 mL CEB and incubated for24 hours at 42° C. under microaerophilic conditions (5% O₂, 10% CO₂, and85% N₂). Following incubation, the strains were pooled in a 25 mL tubeand 3 mL were transferred into a glass tube to be read in aspectrophotometer to determine the concentration of bacteria present inthe culture. After measuring the absorbance (OD), the tube containingthe culture was centrifuged at 3,500×g for 10 min. The supernatant wasdiscarded and the pellet was resuspended in an equal amount ofButterfield's Phosphate Diluent (BPD). The culture was serially dilutedto obtain an inoculum with a desired concentration of 4×10⁵ cfu/mL.Detection of Campylobacter inhibition was assessed by measuring theinhibition zone surrounding the isolate colonies on the plate.

Selection for Motility Enhancement

The GRAS isolates demonstrating motility characteristics and the abilityto inhibit Campylobacter in vitro were further selected for motilityenhanced characteristics. Isolates were incubated in TSB for 24 hours at37° C. Bacteria were transferred into motility test plates (modificationfrom Tittsler and Sandholzer (J. Bacteriol, (1936) 31(6); 575-580) byadding TTC; BBL™ Motility Becton-Dickinson). The plates were incubatedfor 24 hours at 37° C. Migration capability was detected by measuringthe diameter of dispersion from the initial point. If the dispersion wasnot concentric, the measurements were based on the farthest coloniespresent at the extremes. The diameter was measured and recorded in eachpass and, after the last pass the identities of isolates were confirmedusing the Biolog® system. The farthest colony from the center was pickedusing a sterile loop and incubated in TSB at 37° C. overnight. Theprocedure was repeated ten times to further select colonies with bestmigration capability. The bacterial isolates obtained from the lastpassage were identified as motility enhanced (ME) isolates.

The ME isolates demonstrating the greatest motility in vitro (colonieswith the largest diameters from the initiation point on the lastpassage) were used in field trials. For study purposes these strainswere designated as ME isolate 1, 2 or 3.

Animal Studies

In Vivo Testing of Motility Enhanced Isolates against Campylobacter

Three trials were conducted using one-day-old chicks. In each trial 80broilers were randomly allocated into 8 separated floor pens (10birds/pen) for 14 days and one of eight treatments was randomly assignedto each pen. The treatments groups included positive controls(Campylobacter, no probiotic) or probiotics given individually (isolates1, 2 or 3 ME) or in combinations (isolates 1 and 2; 1 and 3; 2 and 3; ora combination of the three isolates ME). At day of hatch, bacterialisolates were administrated to each chick by oral gavage consisting of0.25 mL of TSB broth with approximately 10⁷ cfu/mL. At day seven, allchicks (including the positive control), were challenged via oral gavage(0.25 mL) with a solution of BFD containing approximately 4×10⁵ cfu/mLof a mixture of the four strains of Campylobacter. At 2 weeks of agechicks were euthanized using CO₂ and ceca collected to assessCampylobacter colonization.

In Vivo Comparison of Motility Enhanced Isolate against Campylobacter

The ME probiotic isolate that offered the most consistent reductionduring field testing was chosen to be further tested against itsoriginal strain in an in vivo study. Three trials were conducted. Ineach trial, 30 chicks (n=10/treatment) were randomly allocated into 3separated floor pens for 14 days. The birds were assigned to treatmentgroups: 1) positive controls (Campylobacter, no probiotic); 2) originalstrain of isolate 1 not subjected to ME, also designated originalstrain; or 3) isolate I-ME (pass 10, after enhancement of motilitycharacteristics). At-day-of-hatch probiotic treatment groups wereinoculated by oral gavage (0.25 mL) with approximately 10⁷ cfu/mL of theappropriate bacteria culture. On day seven all groups were challenged byoral gavage with a solution of approximately 4×10⁵ cfu/mL containing thefour strains of Campylobacter. At 2 weeks of age chicks were euthanizedusing CO₂ and ceca collected to assess Campylobacter colonization.

Enumeration of Campylobacter in Cecal Contents

Cecal Campylobacter concentrations were enumerated by the procedure ofCole and co-workers (Poult. Sci. (2006) 85:1570-1575). Briefly, cecafrom each bird were transferred to a sterile plastic bag and thecontents squeezed into 15-mL tubes and serially diluted (1:10) with BPDand inoculated on labeled Campy-Line Agar (CLA) plates. The CLA plateswere incubated for 48 h at 42° C. under microaerophilic conditions.Direct bacterial counts were recorded and converted to cfu/mL of thececal content. Campylobacter colonies were confirmed by latexagglutination test (PANBIO, INC. Columbia, Md.) and further identifiedas Campylobacter jejuni using API® Campy (Biomerieux® Durham, N.C.; ATCC33291 strain used as control).

Statistical Analysis

Data were analyzed using ANOVA with the GLM procedure (SAS Institute,2002). For field trials, the number of Campylobacter colonies werelogarithmically transformed (log 10 cfu/mL) before analysis to achievehomogeneity of variance (Byrd et al., Poult. Sci. (2003) 82:1403-1406).Treatment means were partitioned by LSMEANS analysis (SAS Institute,2002, SAS/STAT® User's guide: Release 9.03 edition. SAS Institute Inc.,Gary, N.C.). A probability of P<0.05 was required for statisticalsignificance.

Results Isolation Studies

In the original probiotic isolation studies, 139 bacteria were isolatedfrom the ceca. Of these, 39 isolates were verified to be GRAS. Six ofthe GRAS isolates were positive for initial motility characteristics.Three out of six were different strains of Bacillus subtilis ssp.

Selection for Motility Enhancement

Five out of the six motile, GRAS isolates demonstrated inhibitionagainst Campylobacter jejuni in vitro (FIG. 3). Not all isolatespresented the same progress during the selection for motilityenhancement. Some of the isolates had their best performance withinpasses rather than the last pass such as isolate 1 (pass 4), isolate 2(pass 9), isolate 3 (pass 6), isolate 4 (pass 1) and isolate 5 (pass 3).In order to standardize the procedure the number of passes was limitedto 10 and the selection was based on the size of the diameter in thelast pass. By using this criterium 3 out of 5 initial isolates presentedthe largest spread in pass 10. For study purposes these strains weredesignated as ME: isolate 1, isolate 2 and isolate 3.

Study 1: In Vivo Testing of Motility Enhanced Isolates againstCampylobacter

Cecal Campylobacter counts for the positive control group averagedapproximately 2×10⁷ cfu/g in the three field trials (FIG. 2). Isolate 1and isolate 2 reduced Campylobacter counts (P<0.05) in cecal contentswhen compared to the positive control in 2 out of 3 trials (FIG. 2).Significant reductions were also found in 1 out of 3 trials for triplecombination (1&2&3) and dual combination (1&2), Trials 1 and 3,respectively. Among them isolate I-ME presented the most consistentreduction, in 2 out of 3 trials, being selected for a further study.

Study 2: In Vivo Comparison of Motility Enhanced Isolate againstCampylobacter

Cecal Campylobacter counts for positive control group averagedapproximately 2×10⁷ cfu/g for all in vivo trials. For all three trialsconducted (FIG. 3), isolate 1-ME consistently reduced Campylobactercecal counts when compared to the positive control group, and resultedin lower Campylobacter cecal counts when compared to the non-motilityenhanced isolate 1. The cecal Campylobacter counts for non-motilityenhanced isolate 1 did not differ from the positive control in alltrials.

Selecting mottle GRAS bacteria from cecal contents and further selectionfor their migration capability resulted in isolation of a probioticbacterium capable of consistently outcompeting Campylobacter for bindingsites in in vivo trials (FIG. 2). Campylobacter ssp. is predominantlyfound in the lower gastrointestinal tract, concentrated in the deepmucus layer of the cecal crypts. One of the possible mechanisms by whichsome of the ME isolates contributed to the reduction in Campylobactercounts may lay in their ability to get to the intestinal niche occupiedby Campylobacter and compete for nutrients and binding sites.

The motility enhanced isolates were all identified as Bacillus subtilis.The genus Bacillus comprises a diverse collection of aerobicendospore-forming, motile bacteria. Bacillus subtilis as well Bacilluslicheniformis species are on the Food and Drug Administration's GRASlist. They have been widely used in the food industry for production ofenzymes such as cellulose and expression of thaumatin II, asweet-tasting protein, non-caloric substitute for sugar and in Japanwere B. subtilis is used to produce fermented soybean products. The useof Bacillus as a probiotic could have more than one mode of action,including competitive exclusion for adhesion sites and production ofantimicrobial agents such as bacteriocins.

We claim:
 1. A method of selecting a first bacterium capable of reducingpathogenic bacterial colonization by a second bacterium in theintestinal tract of a subject comprising selecting the first bacteriumto be capable of migrating at least 0.75 cm from the point ofinoculation on motility agar after incubation for 24 hours at 37° C. orto a diameter of at least 1.5 cm based on the farthest migratingbacterium from the point of inoculation on motility agar afterincubation for 24 hours at 37° C.; and selecting the first bacterium tobe capable of reducing colonization of the subject by the secondbacterium after administration of the first bacterium to the subject. 2.The method of claim 1, further comprising selecting the first bacteriumto be a bacterium Generally Recognized As Safe by the Food and DrugAdministration.
 3. The method of claim 1, further comprising selectingthe first bacterium to be capable of inhibiting the growth ofCampylobacter in vitro.
 4. The method of claim 1, wherein the firstbacterium is selected from Escherichia, Salmonella, Shigella, Bacillus,Clostridium and Bacteriodes.
 5. The method of claim 1, wherein thesecond bacterium comprises a bacterium selected from Campylobacter,Salmonella and Escherichia.
 6. The method of claim 1, further comprisingselecting the first bacterium to be capable of reducing colonization byCampylobacter in the subject.
 7. The method of claim 1, wherein thesubject is poultry, cattle, humans or pigs.
 8. (canceled)
 9. The methodof claim 1, wherein the first bacterium is capable of migrating at least1.5 cm from the point of inoculation on motility agar alter incubationfor 24 hours at 37° C. or to a diameter of at least 3 cm based on thefarthest migrating bacteria from the point of inoculation on motilityagar after incubation for 24 hours at 37° C.
 10. A bacterium selected bythe method of claim
 1. 11. An isolated Bacillus subtilis strain selectedfrom the group consisting of a Bacillus subtilis strain designated asME-1; a Bacillus subtilis strain designated as ME-2; and a Bacillussubtilis strain designated as ME-3.
 12. A composition comprising atleast one of the isolated Bacillus subtilis strains of claim 11, whereinthe composition is a food product, is formulated for addition todrinking water, an aerosol or a sprayable product.
 13. (canceled)
 14. Amethod of inhibiting growth of a second bacterium comprising deliveringa composition comprising one of the strains of claim 11 to a targetregion.
 15. The method of claim 14, wherein the growth of the secondbacterium is inhibited by at least five fold as compared to a control.16. (canceled)
 17. The method of claim 14, wherein the target, region isa solution, area, object, subject or surface suspected of beingcontaminated with or harboring the second bacterium.
 18. (canceled) 19.A method of reducing pathogenic bacterial colonization by a secondbacterium in a subject comprising orally administering a compositioncomprising at least one of the strains of claim 11 to the subject. 20.The method of claim 19, wherein the second bacterium is selected fromCampylobacter, Salmonella and E. coli.
 21. The method of claim 19,wherein the subject is poultry, cattle, humans or pigs.
 22. (canceled)23. The method of claim 19, wherein oral administration is via oralgavage, aerosolization or by addition to food or water.
 24. The methodof claim 19, wherein colonization by the second bacterium is reduced ascompared to control subjects in the gastrointestinal tract of thesubject.
 25. (canceled)
 26. (canceled)
 27. A method of reducingpathogenic bacterial colonization by a second bacterium in a subjectcomprising orally administering a first bacterium to the subject,wherein the first bacterium is capable of migrating at least 0.75 cmfrom the point of inoculation on motility agar after incubation for 24hours at 37° C. or capable of migrating from the point of inoculation toa diameter of at least 1.5 cm based on the farthest migrating bacteriumfrom the point of inoculation on motility agar after incubation for 24hours at 37° C., and wherein administration reduces colonization of thesecond pathogenic bacterium in the gastrointestinal tract of thesubject.
 28. (canceled)
 29. (canceled)